Identification of a Third EspA-binding Protein That Forms Part of the Type III Secretion System of Enterohemorrhagic Escherichia coli

Ku, Chen-Peng; Lio, Chan-Wang Jerry; Wang, Shao–Hung; Lin, Chin-Nan; Syu, Wan-Jr

doi:10.1074/jbc.m807478200

Cited by 15 publications

(18 citation statements)

References 40 publications

(49 reference statements)

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“…The null strains showed defective secretion, indicating that EscL, EscN, and EscQ are essential for T3S (Fig. 1A), in agreement with the observations previously reported for Citrobacter rodentium and EHEC (3,16). Complementation of the null strains with double-hemagglutinin (HA)-tagged EscL, EscN, or EscQ in trans (cloned into pTOPO-2HA [3] by the use of HindIII and XhoI sites with the primer pairs shown in Table 1) restored secretion of the translocators, thus confirming that the deletion mutations in escL, escN, and escQ were nonpolar and that the tagged versions of these proteins were functional (Fig.…”

supporting

confidence: 81%

Quantitative Proteomic Analysis Reveals Formation of an EscL-EscQ-EscN Type III Complex in Enteropathogenic Escherichia coli

et al. 2011

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supporting

confidence: 81%

Quantitative Proteomic Analysis Reveals Formation of an EscL-EscQ-EscN Type III Complex in Enteropathogenic Escherichia coli

et al. 2011

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“…Alternatively, EscK and EscL may directly participate as docking sites for T3S substrates. Indeed, EscL has been shown to bind EspA (82). Moreover, it is interesting that proteins of the SctK family are the less-conserved constituents of the sorting platform structure.…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of EscK (Orf4), a Sorting Platform Component of the Enteropathogenic Escherichia coli Injectisome

et al. 2017

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The type III secretion system (T3SS) is a supramolecular machine used by many bacterial pathogens to translocate effector proteins directly into the eukaryotic host cell cytoplasm. Enteropathogenic Escherichia coli (EPEC) is an important cause of infantile diarrheal disease in underdeveloped countries. EPEC virulence relies on a T3SS encoded within a chromosomal pathogenicity island known as the locus of enterocyte effacement (LEE). In this work, we pursued the functional characterization of the LEE-encoded protein EscK (previously known as Orf4). We provide evidence indicating that EscK is crucial for efficient T3S and belongs to the SctK (OrgA/YscK/MxiK) protein family, whose members have been implicated in the formation of a sorting platform for secretion of T3S substrates. Bacterial fractionation studies showed that EscK localizes to the inner membrane independently of the presence of any other T3SS component. Combining yeast two-hybrid screening and pulldown assays, we identified an interaction between EscK and the C-ring/sorting platform component EscQ. Site-directed mutagenesis of conserved residues revealed amino acids that are critical for EscK function and for its interaction with EscQ. In addition, we found that T3S substrate overproduction is capable of compensating for the absence of EscK. Overall, our data suggest that EscK is a structural component of the EPEC T3SS sorting platform, playing a central role in the recruitment of T3S substrates for boosting the efficiency of the protein translocation process.IMPORTANCE The type III secretion system (T3SS) is an essential virulence determinant for enteropathogenic Escherichia coli (EPEC) colonization of intestinal epithelial cells. Multiple EPEC effector proteins are injected via the T3SS into enterocyte cells, leading to diarrheal disease. The T3SS is encoded within a genomic pathogenicity island termed the locus of enterocyte effacement (LEE). Here we unravel the function of EscK, a previously uncharacterized LEE-encoded protein. We show that EscK is central for T3SS biogenesis and function. EscK forms a protein complex with EscQ, the main component of the cytoplasmic sorting platform, serving as a docking site for T3S substrates. Our results provide a comprehensive functional analysis of an understudied component of T3SSs.

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“…These chaperones increase the stability of EspA and show direct EspA-binding activity (12,13,52). Recently, it has been reported that EscL, in addition to CesAB and CesA2, interacts directly with EspA, enhances the stability of intracellular EspA, and is also essential for the complete secretion of EspA (33). Similarly, we found that two small T3SS molecules, SsaE and SseA, function as chaperones for the Salmonella SPI-2 translocator SseB, an EspA homologue.…”

Section: Discussionmentioning

confidence: 53%

Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by Salmonella Pathogenicity Island 2

Miki¹,

Shibagaki

Danbara³

et al. 2009

J Bacteriol

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The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI-2) is involved in systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. In this study, we investigated the function of SsaE, a small cytoplasmic protein encoded within the SPI-2 locus, which shows structural similarity to the T3SS class V chaperones. An S. enterica serovar Typhimurium ssaE mutant failed to secrete SPI-2 translocator SseB and SPI-2-dependent effector PipB proteins. Coimmunoprecipitation and mass spectrometry analyses using an SsaE-FLAG fusion protein indicated that SsaE interacts with SseB and a putative T3SS-associated ATPase, SsaN. A series of deleted and point-mutated SsaE-FLAG fusion proteins revealed that the C-terminal coiled-coil domain of SsaE is critical for protein-protein interactions. Although SseA was reported to be a chaperone for SseB and to be required for its secretion and stability in the bacterial cytoplasm, an sseA deletion mutant was able to secrete the SseB in vitro when plasmid-derived SseB was overexpressed. In contrast, ssaE mutant strains could not transport SseB extracellularly under the same assay conditions. In addition, an ssaE(I55G) point-mutated strain that expresses the SsaE derivative lacking the ability to form a C-terminal coiled-coil structure showed attenuated virulence comparable to that of an SPI-2 T3SS null mutant, suggesting that the coiled-coil interaction of SsaE is absolutely essential for the functional SPI-2 T3SS and for Salmonella virulence. Based on these findings, we propose that SsaE recognizes translocator SseB and controls its secretion via SPI-2 type III secretion machinery.

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Identification of a Third EspA-binding Protein That Forms Part of the Type III Secretion System of Enterohemorrhagic Escherichia coli

Cited by 15 publications

References 40 publications

Quantitative Proteomic Analysis Reveals Formation of an EscL-EscQ-EscN Type III Complex in Enteropathogenic Escherichia coli

Quantitative Proteomic Analysis Reveals Formation of an EscL-EscQ-EscN Type III Complex in Enteropathogenic Escherichia coli

Functional Characterization of EscK (Orf4), a Sorting Platform Component of the Enteropathogenic Escherichia coli Injectisome

Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by Salmonella Pathogenicity Island 2

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